It is widely held that the establishment and course of HIV infection is determined by the interplay between viral replication and humoral responses to viral envelope trimers that mediate host cell attachment and entry. Accordingly, intensive efforts have been directed towards understanding the structural, functional and antigenic characteristics of HIV trimers. Such information promises to reveal insights for developing antiviral countermeasures, including envelope-targeted antiviral agents and vaccines based on anti-envelope antibodies. The nature of HIV envelope trimers has been examined primarily in the context of free virions. However, several lines of evidence suggest that HIV envelope trimers on virions transition through a series of conformations in solution and/or as they engage up to three CD4 receptor molecules during viral attachment. This in turns suggests certain testable hypotheses concerning the nature of viral trimers. One that will be tested in this project holds that the flexibility and antigenicity of trimers progressively changes as trimers become progressively saturated with CD4. A related hypothesis, also explored in this project, is that certain conformations allow combinations of neutralizing antibodies and/or both neutralizing and non-neutralizing antibodies to bind a single trimer. Our preliminary studies suggest that the latter situation frequently occurs on virions, whereas data from virus capture assays of immunochemical studies with purified soluble trimers suggest that neutralizing epitopes are expressed on native trimers in the absence of non-neutralizing epitopes. Accurate evaluations of these hypotheses demands a means to interrogate virions as they naturally exist in solution, without extensive technical manipulation. Previously we reported a single molecule approach based on the use of fluorescence correlation spectroscopy (FCS) to characterize epitope exposures on free virions with all reactants (e.g. antibodies, soluble CD4) continuously in solution. More recently, we have expanded this approach such that we can probe virions simultaneously with multiple antibodies in combination with fluorescence resonance energy transfer (FRET). This novel approach detects multiple epitope exposures on a single virion trimer. It concurrently reveals conformational dynamics as a function of FRET between two epitope probes. Most importantly, such measures are accomplished using our approach without genetic or external perturbations of virions. Thus, we can collect unprecedented information regarding the structural and antigenic dynamics of the HIV envelope, which should provide novel insights for the generation of antiviral agents and anti-HIV immunity. We propose three aims: Aim 1) To characterize the exposure of multiple epitopes on single, virion-associated HIV trimers. Aim 2) To examine the conformational dynamics of gp120 on HIV virions. Aim 3) To characterize conformational flexibility before and during ligand binding to soluble cleaved trimers.